Bacillus Pumilus Bilirubin Oxidase and Applications Thereof

ABSTRACT

The present invention relates to a novel  Bacillus pumilus  bilirubin oxidase, to the method for preparing same and also to the use thereof in particular for assaying bilirubin and for using enzymatic biofuel cells.

The present invention relates to a novel bilirubin oxidase, to themethod for preparing same and also to the use thereof in particular forassaying bilirubin and for the use of enzymatic biofuel cells usingoxygen as fuel.

Bilirubin oxidase or BOD (E.C. 1.3.3.5.) is an enzyme which catalysesthe reaction for oxidation of bilirubin to biliverdin:

bilirubin+½ O₂→biliverdin+H₂O

BOD has four sites for binding to copper atoms; these four copper atomsare necessary for correct activity of the enzyme; it has in fact beenshown that the absence of a copper in the CotA protein of Bacillussubtilis (a protein with bilirubin oxidase activity sold, as BOD, by thecompany Genzyme Diagnostics) is sufficient to reduce the activity of theenzyme (table 3 of the article by Durao et al., in J Biol Inorg Chem.2008 February; 13(2): 183-93).

Bilirubin is a yellow substance formed in the blood by the decompositionof haemoglobin; it is one of the main pigments produced in the liver.

BOD is of interest for various applications, such as the assaying ofbilirubin, making it possible, for example, to diagnose excess bilirubinin the blood; it can also be used to prepare enzymatic biofuel cellswhere it will capture cathode electrons, reducing oxygen to water (seethe schematic representation of an enzymatic biofuel cell where the BODis attached in a redox polymer to the cathode, in FIG. 1A) or as anoxygen biosensor.

There are many sources of BOD; this enzyme can be produced frommicroorganisms such as those of the Bacillus genus [Bacillus subtilis,the CotA of which has a bilirubin oxidase activity, see Sakasegawe etal. 2006 Applied and Environmental Microbiology 72, No. 1, 972-975;Bacillus licheniformis (U.S. Pat. No. 4,770,997)], or from mycetes,among which, those of the genus Penicillium [Penicillium janthinellum(patent application EP 0 295 101)], Trachyderma (U.S. Pat. No.4,600,689), Myrothecium (Tanaka et al. 1982 Agric. Biol. Chem. 46,2499-2503) or else Schizophyllum, Coprinus, Trametes, Coriolus,Pholiota, Pleurotus, Lenzites or Fomitopsis (U.S. Pat. No. 4,677,062).

This enzyme can also be extracted from plants such as of the typeAlfalfa (U.S. Pat. No. 5,624,811), Solanaceae, Musaceae and Liliaceae(EP 0 140 004) or else Compositae, such as the artichoke (EP 0 247 846).

Among these enzymes, the BODs having the most advantageous enzymaticproperties, in particular activity and stability, have been selected tobe marketed; they are Bacillus subtilis CotA having bilirubin oxidaseactivity (it is sold as BOD by the company Genzyme Diagnostics and willsubsequently be denoted BOD) and Myrothecium verrucaria BOD (sold by thecompanies Sigma-Aldrich and Amano).

The inventors have now identified a novel BOD produced by Bacilluspumilus which is more active and/or more stable than the commerciallyavailable BODs; they have also developed a method for preparing thisnovel BOD which is simpler and faster than those used to date for theother known BODs.

According to a first subject, the invention relates to the wild-type BODof Bacillus pumilus; in particular, the bilirubin oxidase, in particularthe purified bilirubin oxidase (purity >95%) according to the inventionhas a percentage identity of at least 80%, and by order of increasingpreference at least 85%, 90%, 95%, 97%, 98% and 99% identity, withrespect to the wild-type BOD of Bacillus pumilus of SEQ ID No. 2; itcatalyses the reaction for oxidation of bilirubin to biliverdin and isbound to four copper atoms.

SEQ ID No. 2 corresponds to the wild-type BOD of the Bacillus pumilusstrain SAFR032. By way of example, the present invention also relates tothe wild-type BODs of other Bacillus pumilus strains, for instance theBOD of the ATCC 7061 strain of SEQ ID No. 6, which has a percentageidentity of 98% with the BOD of SEQ ID No. 2; the preferred BODaccording to the invention is the wild-type BOD of the Bacillus pumilusstrain SAFR032 of SEQ ID No. 2.

The identity of a sequence with respect to the sequence of the wild-typeBOD of Bacillus pumilus (SEQ ID No. 2) as reference sequence is assessedaccording to the percentage of amino acid residues which are identical,when the two sequences are aligned, so as to obtain the maximumcorrespondence between them.

Protein sequences predicted from the systematic sequencing of theBacillus pumilus genome are described in the UniProt database (accessionnumber A8FAG9 “Outer Spore Coat Protein A” of 13 Nov. 2007 and accessionnumber B4AIB1 “Spore Coat Protein A” of 23 Sep. 2008); it should beunderlined that the information presented in the UniProt database ispredictive and putative, it does not result from the experimentalisolation and characterization of Bacillus pumilus proteins. Inaddition, the indications appearing in this database did not make itpossible to predict any BOD activity for these proteins, since, amongthe various CotA characterized to date from the organisms B. subtilis,B. licheniformis (Koschorreck, K., et al., Cloning and characterizationof a new laccase from Bacillus licheniformis catalysing dimerization ofphenolic acids. Appl Microbiol Biotechnol, 2008. 79(2): p. 217-24;Koschorreck, K., R. D. Schmid, and V. B. Urlacher, Improving thefunctional expression of a Bacillus licheniformis laccase by random andsite-directed mutagenesis. BMC Biotechnol, 2009. 9: p. 12), B.halodurans, and B. HR03, before the BOD of B. pumilus, only that of B.subtilis has been characterized as a BOD, the others being laccases(enzymes having a weak tetrapyrrole-oxidizing activity, unlike BODS).

The percentage identity can be calculated by those skilled in the artusing a sequence comparison computer program such as, for example, thatof the BLAST series (Altschul et al., NAR, 25, 3389-3402). The BLASTprograms are implemented on the window of comparison consisting of theentire SEQ ID No. 2 indicated as reference sequence.

A peptide having an amino acid sequence having at least X % identitywith a reference sequence is defined, in the present invention, as apeptide of which the sequence can include up to 100-X modifications per100 amino acids of the reference sequence, while retaining thefunctional properties of said reference peptide, in the case in pointits bilirubin oxidation enzymatic activity. For the purpose of thepresent invention, the term “modification” includes consecutive ordispersed deletions, substitutions or insertions of amino acids in thereference sequence.

The novel BOD according to the invention has improved propertiescompared with the commercially available BODs derived from Myrotheciumverrucaria or Bacillus subtilis.

In particular, the Bacillus pumilus BOD has better enzymatic properties(activity, catalytic efficiency k_(cat) and affinity of the substratefor the enzyme K_(M)) with respect to catalysis of the oxidation of2,2′-azinobis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), than theBODs of Myrothecium verrucaria or of Bacillus subtilis.

The enzymatic properties can be determined as described in part 4 of theexample which follows.

Table I below gives the catalytic efficiency k_(cat), i.e. the number ofmolecules of substrate converted to product per molecule of enzyme andper unit time, and the Michaelis constant K_(M) which represents theaffinity of the substrate (ABTS) for the BODs of B. subtilis and of B.pumilus.

The enzymatic properties of the BODs of B. pumilus and of B. subtiliscan easily be compared since these two enzymes have very similar optimalconditions for use: pH between 3 and 4 and temperature between 75 and80° C.

TABLE I Enzymatic properties of the BODs of B. pumilus and of B.subtilis BOD k_(cat) (for ABTS) K_(M) B. pumilus 391.3 s⁻¹ 31.7 μM B.subtilis 322 s⁻¹ 124 μM

The enzymatic properties described for the M. verrucaria BOD by Kataokaet al. (2005, Protein Expression and Purification, 41, 77-83) at pH 6.5are a k_(cat) of 115 s⁻¹ and a K_(M) of 250 μM. Moreover, Sakurai et al.(2008, Biochemical and Biophysical Research communication, 371, 416-419)have determined the specific activity of the M. verrucaria BOD for ABTS,which is 106 U/mg, the specific activity of the BOD according to theinvention itself being 375 U/mg. In addition, the B. pumilus BOD hasvery good heat stability and good bilirubin oxidation enzymaticproperties.

The present invention also relates to a nucleic acid molecule encodingthe BOD according to the invention; it is preferably a nucleic acidmolecule having a sequence chosen from SEQ ID. No. 1 encoding thewild-type BOD of Bacillus pumilus SARF-032, SEQ ID No. 5 encoding thewild-type BOD of Bacillus pumilus ATCC 7061 or else SEQ ID No. 7 whichcorresponds to the sequence of the wild-type BOD of Bacillus pumilusSARF-032 which has been modified in order to improve the expressionthereof by the yeast Pichia pastoris. The nucleic acid molecule encodingthe BOD according to the invention can be cloned into an expressionvector, such as a plasmid, and then used to transform a suitable host,such as a bacterium, a yeast or else a cell culture.

The term “expression vector” is intended to mean a vector which has aregion allowing the insertion of a coding nucleotide sequence betweenthe signals essential for its expression, in particular a promoter(constitutive or inducible), a ribosome-binding site, a transcriptionstop signal and, optionally, a selectable marker, such as a gene forresistance to an antibiotic.

The present invention also relates to an expression vector comprisingsaid nucleic acid molecule and to a host cell transformed with saidexpression vector and expressing a BOD according to the invention.

The introduction of the expression vector into the host cell can becarried out by any method known to those skilled in the art, inparticular by a modification of the membrane permeability of the hostcell, for example in the presence of calcium ions, or byelectroporation. After culture of the host cells transformed so as toexpress the BOD according to the invention, said cells can be recoveredby centrifugation, and lysed in order to release the enzymes, includingsaid BOD according to the invention.

If Escherichia coli is the host microorganism, the plasmids which can beused are in particular the plasmids pBluescript, pUC18, pET, pGEX, pGS,pMAL-c2, or the like.

According to a preferred method for preparing the BOD according to theinvention, the BOD is expressed by an E. coli bacterium transformed witha pET2la expression vector encoding an enzyme joined to a 6HIS tag inthe C-terminal position.

This method, illustrated in the experimental section which follows(section 3), is advantageous owing to its rapidity and its simplicity;this is because the induction of the Bacillus pumilus BOD expression inthe E. coli bacterium takes place in 4 to 24 hours, whereas theproduction of BOD derived from Myrothecium verrucaria requires inductionperiods that can reach 5 days (Kataoka et al. Biochemistry. 2005 May 10;44(18):7004-12; Kataoka et al., Biochem Biophys Res Commun. 2008 Jul. 4;371(3):416-9; Kataoka et al. K. Protein Expr Purif. 2005 May;41(1):77-83).

In addition, the 6HIS tag makes it possible to purify the Bacilluspumilus BOD by affinity chromatography on a nickel resin in a singlestep so as to obtain a pure enzyme; the small size of the tag (6 aminoacids) makes it possible to do away with eliminating it since it doesnot significantly disturb the activity of the enzyme. By way ofcomparison, the purification of the Myrothecium verrucaria and B.subtilis BODS which is described, respectively, in the articles byKataoka et al. (see above) and Durao et al. (J Biol Inorg Chem. 2008February; 13(2):183-93) require several chromatography steps.

The rapidity and the simplicity of this method therefore representconsiderable advantages compared with the methods for preparing enzymesthat are currently commercially available.

Those skilled in the art will select the host cell according to theexpression vector used.

Preferably, when the pET21a expression vector is used, a host cellexpressing the T7 RNA polymerase, such as the E. coli strains BL₂₁ DE3,BL₂₁-SI, BL₂₁ pLys, Novablue(DE3) or BL₂₁ Star, will be selected.Preferably, the Bacillus pumilus BOD according to the invention isproduced in an Escherichia coli BL₂₁ Star strain; the nucleic acidmolecule which encodes it is obtained by PCR with the primers of SEQ IDNos. 3 and 4 and cloned into the pET2la vector so as to give thetransformed vector pFD1. The BOD thus produced is then purified, afterlysis of the bacteria, by affinity chromatography.

According to another advantageous variant of the invention, the BODaccording to the invention is produced by the Pichia pastoris yeast.

In order to allow the overproduction and the secretion of the BOD intothe culture medium of the Pichia pastoris yeast, the gene encoding theBOD, in particular chosen from the sequences SEQ ID No. 1, 5 or 7,preferably SEQ ID No. 7, is introduced by homologous recombination intothe yeast genome, at the level of the AOX1 gene. For this, the pFD2plasmid, once linearized by digestion with the pmel enzyme, isintroduced into the yeast by electroporation, and the positive clonesare selected on YPD+agar medium containing zeocin at 100 μg/ml. Apreculture of 200 ml of YPD medium supplemented with zeocin (100 μg/ml)is inoculated using an isolated clone on a Petri dish.

After shaking at 220 rpm ‘overnight at 30° C., this pre-culture is thencentrifuged for 10 min at 4000 rpm and the pellet is taken up in 200 mlof sterile water in order to remove any presence of glucose. After asecond centrifugation, a 2 L culture in MMH medium containing 1 mM ofCuSO₄ in a 5 L Erlenmeyer flask is then inoculated with this pellet. Theyeasts are incubated at 25° C. with shaking (220 rpm) for 2 hours,before the addition of 0.5% of methanol in order to initiate theinduction. This induction step will be repeated for 5 days in order toobtain the maximum amount of enzymes.

In order to implement this method, the following material can be used,without being limiting in nature:

-   -   vector for expression in Pichia pastoris (pFD2): pPICZa plasmid        containing the DNA sequence encoding the Bacillus pumilus BOD,        preferably optimized (SEQ ID No. 7), in frame with the        Saccharomyces cerevisiae α-factor secretion factor and        containing the methanol-inducible AOX1 promoter;    -   Pichia pastoris yeast strain GS115 used for producing bilirubin        oxidase after integration of the cassette derived from the PFD2        vector containing the AOX1 promoter, the α-factor signal peptide        and the DNA sequence encoding the Bacillus pumilus BOD;    -   culture media:

YPD Rich Medium (for Yeast):

-   -   1% yeast extract    -   2% bactopeptone    -   2% glucose    -   pH not adjusted, autoclaved for 20 min at 120° C.

MMH Minimum Medium (for Yeast):

-   -   1.34% yeast nitrogen base    -   1% Casamino acid    -   0.4% histidine    -   4×10⁻⁵% biotin    -   pH not adjusted, autoclaved for 20 min at 120° C.

LB Rich Medium (for Bacterium):

-   -   10 g/l tryptone    -   5 g/l yeast extract    -   5 g/l NaCl    -   Distilled H₂O qs 11    -   pH not adjusted, autoclaved for 20 min at 120° C.

The present invention also relates to a method for preparing a BODaccording to the invention, comprising the steps of:

-   -   a) preparing host cells expressing the BOD according to the        invention;    -   b) culturing the host cells prepared in step a);    -   c) lysing the host cells;    -   d) treating the lysate obtained in step c) by affinity        chromatography;    -   e) recovering said purified BOD.

According to one preferred embodiment, the method according to theinvention is such that:

-   -   the Escherichia coli BL₂₁ Star strain transformed with the pFD1        vector is prepared in step a);    -   the culture carried out in step b) is a liquid-phase culture,        with shaking, under anaerobic conditions for a period of 4 to 30        h, preferably 24 h, at a temperature between 18 and 37° C.,        preferably 20° C., during which the BOD expression is induced by        adding isopropyl-β-D-1-thiogalactopyranoside (IPTG). When the        method is implemented according to these preferred conditions,        it allows the production of the BOD with a short induction time,        of about 24 hours; the purification of the BOD is carried out in        a single affinity chromatography step and the BOD thus produced        indeed comprises the four copper atoms necessary for its        activity (see part 5 of the example).

It is also possible to produce a BOD in the presence of denaturingagents such as urea, guanidinium chloride, SDS, triton, etc., the BODthus produced will then be devoid of copper and may be activated byadding copper ions.

The invention also relates to the use of the Bacillus pumilus BODaccording to the invention for assaying bilirubin in solution, i.e.measuring the bilirubin concentration in a sample, in particular abiological sample.

The term “biological sample” is intended to mean a biological fluid,such as blood, serum, lymph, bile, urine, cerebrospinal fluid, sweat,etc.

The presence of bilirubin in the organism is normal, it comes from thedegradation of haemoglobin and approximately 200 to 230 mg of bilirubinare formed per day in a healthy adult. In an individual in good health,the bilirubin is taken up by the liver and then degraded; itsconcentration should not therefore exceed certain thresholds, and theassaying of bilirubin is useful for detecting pathological conditionssuch as:

-   -   cases of substantial haemolysis: congenital or acquired        haemolytic anaemia, drug-related, toxic or infectious        haemolysis, transfusion accidents, etc.;    -   insufficient hepatic uptakes or conjugations: Gilbert disease,        Criggler-Najjar disease, the taking of rifampicin        (antitubercular antibiotic);    -   hepatic and biliary conditions: the various types of hepatitis        (viral, toxic, drug-related), the various types of cirrhosis,        rare metabolic abnormalities (Rotor's disease, Dubin-Johnson        disease);    -   biliary conditions;    -   biliary lithiasis;    -   pancreatitis;    -   pancreatic or bile duct cancer.

The present invention thus relates to the use of the BOD according tothe present invention for measuring the bilirubin concentration in aliquid sample, in particular a biological sample.

According to a first variant, the principle of the assaying of bilirubinwith BOD is based on measuring the change in colour of the sample causedby the degradation of the bilirubin.

Bilirubin exhibits a light absorption peak (λ_(max)) at 440 nm; when itis enzymatically degraded by a BOD, the absorbance at λ_(max) of thesample in which it is present decreases; this decrease makes it possibleto quantify the bilirubin initially present in the sample by comparisonwith the decrease in absorbance at 440 nm of calibration solutionscontaining known bilirubin contents measured under the same experimentalconditions.

The present invention also relates to a kit for assaying bilirubin insolution, characterized in that it comprises a BOD according to theinvention.

Typically, the assaying kit also contains the reagents necessary forcarrying out the bilirubin assay test, in particular:

-   -   the buffers;    -   the standard solutions of bilirubin for producing calibration        curves, and    -   the set of instructions necessary for carrying out the assay.

The present invention also relates to a method for assaying thebilirubin in solution in a liquid sample, characterized in that itcomprises the following steps:

-   -   a) measuring the absorbance at λ_(max)=440 nm of said liquid        sample before enzymatic reaction;    -   b) introducing a BOD according to the invention into said liquid        sample;    -   c) measuring the absorbance at λ_(max)=440 nm of said liquid        sample after enzymatic reaction;    -   d) calculating the difference in absorbances measured in        steps a) and c) and comparing this difference with differences        in absorbances measured for standard solutions having a known        bilirubin content;    -   e) determining the initial concentration of bilirubin of said        liquid sample.

According to another variant, the assaying of the bilirubin in a liquidsample is carried out by means of an electrochemical method which usesan electrode including the BOD according to the invention. Thus, thepresent invention also relates to BOD electrodes comprising a conductivematerial, such as a conductive metal, in particular platinum, copper,silver, aluminium, gold or steel, or carbon, for instance vitreouscarbon, carbon fibres, fibres of carbon nanotubes or alternatively whichare made of diamond, etc., said conductive material being coated with adeposit comprising at least one BOD according to the invention, it alsobeing possible for said deposit to comprise a redox polymer in order toimprove the electrical conduction between the enzyme and the electrodeand also the stability of the system.

The redox polymer can, for example, be chosen from ferrocene-based,osmium-based and ruthenium-based polymers and conducting polymers suchas, for example, polypyrrole and polyanaline.

The methods for immobilizing the BOD on said conductive material can bechosen from the conventional methods available to those skilled in theart, which comprise, in particular, embedding of the BOD in a polymermatrix, adsorption of the BOD at the surface of the polymer membrane,attachment by covalent bonding, electrodeposition (Gao et al., Chem.Int. ED. 2002, 41, No. 5, 810-813) or else the technique described inUnited States patent application US 2009/0053582. According to oneembodiment variant, the BOD electrode on which the BOD is immobilized isalso coated with a membrane which prevents the detachment of said enzymefrom the electrode. According to the applications envisaged, saidmembrane can be constituted of nafion, of cellulose or of any otherbiocompatible material, i.e. material compatible with a physiologicalenvironment.

The present invention thus also relates to a bilirubin biosensorconstituted of a BOD electrode according to the invention. Generally, abiosensor consists of an electrode on which a bioreceptor capable ofrecognizing a biological target is immobilized; the binding of thebiological target to the bioreceptor results in physicochemicalmodifications of the membrane and the production of an electrical signalby an electro-chemical (amperometric, potentiometric, conductometric,etc.) transducer joined to the electrode. In the present case, thebiosensor is a BOD according to the invention and the biological targetis bilirubin.

The present invention also relates to a method for assaying bilirubin insolution in a liquid sample with a bilirubin biosensor according to theinvention. According to one variant of use of the bilirubin biosensor,the latter is implanted under the skin of an individual and makes itpossible to record the bilirubin concentration in the blood of saidindividual.

The present invention also relates to an oxygen sensor constituted of anelectrode according to the invention. The BOD electrode according to theinvention can also be advantageously used as a cathode in an enzymaticbiofuel cell; FIG. 1A represents schematically the operating principlefor an enzymatic biofuel cell. The enzymatic biofuel cells according tothe invention are devices comprising a BOD electrode as a cathode and ananode where a substrate oxidation reaction takes place (catalysed by the“enzyme X”); by way of illustration, the substrate may be glucose andthe “enzyme X” glucose oxidase; such a cell is of particular interestwhen the biofuel cell is implanted in an individual for a medicalapplication. The substrate can also be chosen, for example, fromnitrites, nitrates, sulphides, urates, ascorbates, glutamates,pyruvates, lactates, cellulose, etc., if an application in depollutionis envisaged; the choice of the enzyme will then be made according tothe substrate to be degraded; by way of example, the following enzymescan be used, the type of substrate that they can degrade is mentionedbetween parentheses: glucose oxidase (glucose or any sugars that areoxidized by this enzyme), lactate oxidase (lactate), pyruvate oxidase(pyruvate), alcohol oxidase (alcohol), cholesterol oxidase(cholesterol), glutamate oxidase (glutamate), pyranose oxidase(pyranose), choline oxidase (choline), cellobiose dehydrogenase(celloboise), glucose dehydrogenase (glucose or any sugars that areoxidized by this enzyme), pyranose dehydrogenase (pyranose), fructosedehydrogenase (fructose), aldehyde oxidase (aldehyde), gluconolactoneoxidase (gluconolactone), alcohol dehydrogenase (alcohol), ascorbateoxidase (oxygen or ascorbate) or else sulphide dioxygenase (sulphide).The concomitant oxidation and reduction process at the electrodes of thebiofuel cell produces an electric current.

FIG. 1B illustrates more specifically a glucose-based enzymatic biofuelcell; such an enzymatic biofuel cell consists of two electrodes modifiedby the immobilization of enzymes. A glucose oxidase (GOx) is attached tothe anode (1) by means of a conducting polymer “I” and a bilirubinoxidase (BOD) is attached to the cathode (2) by means of a conductingpolymer “II”. In operating mode, at the anode, the electrons aretransferred from the glucose present in the physiological fluid to theGOx, then from the GOx to the conducting polymer “I” and from theconducting polymer “I” to the anode. At the cathode, the electrons aretransferred from the cathode to the conducting polymer “II”, then to theBOD and, finally, from the BOD to the oxygen present in thephysiological fluid.

It should be noted that a biofuel cell can also optionally operate bymodifying the electrodes with their respective enzymes and addingsoluble mediators, such as ferrocenemethanol for the anode and potassiumferricyanide for the cathode, and adding, as appropriate, a membraneseparating the anode and the cathode.

According to another aspect, the present invention relates to the use ofa BOD according to the invention for degrading the bilirubin present ina sample, in particular a biological sample. This is because thepresence of bilirubin in a sample is capable of distorting the detectionof other substances (such as blood glucose or blood cholesterol) inparticular when these other substances are detected by a colorimetricmethod.

Generally, the BODS according to the invention have many industrialapplications, in particular in the textile and paper industries and inthe food sector, in order, for example, to improve the stability and/orthe quality of foods, such as beverages, or else foods containingvegetable oils, by deoxygenation.

More specifically, the BODS can be used for applications related todepollution; by way of example, mention may be made of the discolorationor the detoxification of wastewater and the degradation of xenobiotics;as organic synthesis reactants; for the preparation of antimicrobialcompositions; for the production of articles made of wood and of cartonswhich have been detoxified or else for the production of detergent(Morozova at al. Biochemistry (Mosc.) 2007 October; 72(10):1136-50) andfor the discoloration of dyes used in industrial media.

The BOD according to the invention can also be used for dimerizingphenolic acid (Koschorreck, K., et al. 2008. Appl Microbiol Biotechnol(2008) 79:217-224) and thus is of interest in the synthesis of pigmentsand dyes used in textile and food applications (R. Mustafa et al. FoodResearch International. Volume 38, Issues 8-9, October-November 2005,pages 995-1000); this dimerization reaction can also be used for thepreparation of antioxidant compounds, for instance ferulic acid dimers(Garcia-Conesa M T, et al. Redox Rep. 1997 Oct-December; 3(5-6):319-23).

The BOD according to the invention can also be used as a reactant in acomposition for the oxidation dyeing of keratin fibres, such as ahair-dyeing composition, comprising, in a medium suitable for dyeing, atleast one oxidation base, a BOD according to the invention and,optionally, a donor for said BOD (such as a substrate, for instancebilirubin). The various ingredients, other than the BOD, that can beused in said composition are described in international application WO99/15138; by way of example, the oxidation base(s) can be chosen frompara-phenylene-diamines, double bases, para-aminophenols,ortho-aminophenols and heterocyclic oxidation bases.

The BOD according to the invention can advantageously be used fortreating wood pulp for its action on lignin degradation and/or forproducing a paper which has a better wet strength (see internationalapplication WO 00/68500).

In addition to the above arrangements, the invention also comprisesother arrangements which will emerge from the description that follows,which refer to exemplary embodiments of the present invention, and alsoto the appended figures in which:

FIGURES

FIG. 1A represents schematically the operating principle for anenzymatic biofuel cell; FIG. 1B represents a glucose-based enzymaticbiofuel cell.

FIG. 2 represents the plasmid map of the pFD1 vector.

FIG. 3 is a graph illustrating the specific activity, in U/mg, of theBacillus pumilus BOD as a function of the ABTS concentration at 37° C.

FIG. 4 is a graphic representation of the Michaelis-Menten equation (k₅₅in s⁻¹ as a function of the unconjugated bilirubin concentration) forthe Bacillus pumilus BOD at 37° C.

FIG. 5 represents the catalytic activity for oxidation of conjugatedbilirubin by the Bacillus pumilus BOD at 37° C. in a 50 mMcitrate/phosphate buffer, pH 4.8.

FIG. 6 represents the catalytic activity for oxidation of syringaldazine(SGZ) by the Bacillus pumilus BOD at 37° C. in a 50 mM citrate/phosphatebuffer, pH 6.2.

FIG. 7 represents the catalytic activity for oxidation of DMP by theBacillus pumilus BOD at 37° C. in a 50 mM citrate/phosphate buffer, pH6.8.

FIG. 8 represents the relative activity of the Bacillus pumilus BOD withrespect to various substrates as a function of the pH.

FIGS. 9A and 9B are graphs representing the stability as a function ofpH of the Bacillus pumilus BOD on ABTS oxidation at 4° C.

FIG. 10 is a histogram representing ABTS oxidation as relative activityby the Bacillus pumilus BOD as a function of temperature.

FIGS. 11A and 11B represent graphically the stability (expressed asspecific activity and as relative activity on ABTS oxidation) of theBacillus pumilus BOD as a function of enzyme incubation time at 80° C.

FIGS. 12A and 12B represent graphically the activity (expressed asspecific activity and as relative activity on ABTS oxidation) of theBacillus pumilus BOD as a function of urea concentration at 25° C. or37° C. in a 100 mM citrate/phosphate buffer, pH 3.

FIG. 13 represents the relative activity of the oxidation of SGZ by theBacillus pumilus BOD as a function of NaCl concentration.

FIG. 14 represents the discoloration of RBBR at 80 mg.l⁻¹ by theBacillus pumilus BOD at 37° C. in a 50 mM potassium phosphate buffer, pH6, in the presence or absence of 10 μM ABTS.

EXAMPLE

1. Materials

1.1 Escherichia coli Bacterial Strains

DH₅α: supE44, ΔlacU169, (Φ80 lacZDM15), hsdR17, recA1, endA1, gyrA96,thi-1, relA1 (Hanahan, 1983).

This strain is used to amplify plasmids during the steps forconstructing the protein expression vectors.

BL₂₁ Star: F-ompT hsdSB(rB-, mB-) gal dcm rnel31 (DE3) (Invitrogen).

This strain is used to produce the Bacillus pumilus BOD in Erlenmeyerflasks.

This strain is then transformed with the pFD1 plasmid which contains theDNA sequence encoding the Bacillus pumilus BOD under the control of theT7 promoter in the pET2la vector.

1.2 Vector

pFD1: pET2la plasmid containing the nucleic acid sequence SEQ ID No. 1encoding the Bacillus pumilus BOD cloned in-frame with the 6×His tag inthe C-terminal position.

The plasmid map of the pFD1 vector is represented in FIG. 2.

1.3 Culture Medium

LB Rich Medium:

10 g/l tryptone

5 g/l yeast extract

5 g/l NaCl

Distilled H₂O qs 1 L

pH not adjusted, autoclaved for 50 min at 1 bar.

2. Genetic Engineering Techniques

2.1 Transformation of Supercompetent Bacteria

Supercompetent DH_(5α) bacteria are prepared using the SEM method(Simple and Efficient Method) according to the protocol described byInoue et al. (Inoue et al. 1990, Gene 96:23-28).

2.2 DNA Preparation

A plasmid DNA purification kit (Quiagen) is used for the DNApreparations in small and large amounts.

2.3 Double-Stranded DNA Sequencing

The double-stranded DNA is sequenced. The sequencing reactions arecarried out with the BigDye Terminator v1.1 or v3.1 sequencing kit. Thereagent contains the 4 ddNTPs with various fluorescent labels (BigDyeTerminators), the AmpliTaq DNA polymerase, and all the other componentsnecessary for the reaction. The extension products should be purifiedbefore being passed through an ABI 3130x1 sequencer, in order to removethe unincorporated labels, the salts and the other contaminants.

2.4 Construction of the BOD Expression Vector

The PCR is carried out with the Phusion HF DNA polymerase on the genomicDNA of the Bacillus pumilus bacterium, strain SAFR-032. The twooligodeoxyribo-nucleotides, complementary to the 3′ and 5′ ends of theDNA sequence of the gene encoding the Bacillus pumilus BOD (SEQ ID No. 3and 4) will be used as primers for the DNA synthesis.

The amplified product and also the pET21a plasmid are then treated withthe two restriction enzymes BamHl and Xhol, the recognition sequences ofwhich have been introduced into the sense oligonucleotide for BamHl andthe antisense oligonucleotide for Xhol, respectively denoted SEQ ID No.3 and 4. The digestion products are gel-purified with the “Nucleospin®”kit (NucleoSpin® Extract II, Clontech Laboratories, Inc.) and the BODgene is then ligated into the plasmid by coincubation with T4 DNA ligaseat 37° C. overnight. The newly formed plasmids are then selected andamplified by transformation of DH5α bacteria on a plate containingampicillin.

TABLE II List of primers used Primer name Sequence SEQ ID No.B. pumilus_S_BamH1 CATGGATCCATGAACCTAGAAAAATTTGTTGACGAG 3B. pumilus_AS_Xhol TACCTCGAGAATAATATCCATCGGCCTCATCATGTC 4

3. Production, Purification and Characterization of the Bacillus pumilusBilirubin Oxidase Enzyme

3.1 Production of Wild-Type BOD Enzymes

The BOD enzyme is produced in the E. coli BL₂₁ star strain by the pET21arecombinant plasmid carrying the sequence encoding wild-type BOD. A 50ml preculture of LB medium supplemented with ampicillin (150 mg/l) (LBA)and 0.25 mM CuSO₄ is inoculated with a clone isolated on an LB agarplate supplemented with ampicillin (100 mg/l), and left shaking, at 220rpm, overnight at 37° C. Two litres of LBA medium containing 0.25 mMCuSO₄, in a 5 L Erlenmeyer flask, are then inoculated at 1/100^(th). Thelatter is incubated at 37° C. with shaking (220 rpm) until anOD_(600 nm) of between 0.8 and 1 OD_(600 nm)/ml is obtained. The cultureis then induced with 200 μM of IPTG and left shaking (180-220 rpm) at25° C. for 4 hours. The cells are then transferred into a sterile 2 LSchott bottle containing a magnetic bar, so as to continue, for 20hours, the culture and the protein induction with shaking underanaerobic conditions in order to increase the incorporation of copperinto the bacteria. The cells harvested by centrifugation (4000 g, 4° C.)are washed in water and stored at −20° C. It is important to emphasizethat the induction of the expression of this BOD in the E. colibacterium is carried out in only 24 hours; this represents aconsiderable advantage compared with the protocols for induction of thecommercial enzymes currently available. This is because the productionof BODS derived from Myrothecium verrucaria can require inductionperiods of up to 5 days.

3.2 Purification of Wild-Type BOD Enzymes

3.2.1 Rupture of Cells and Treatment with DNase I

The cell pellet, derived from two litres of culture, is taken up in 40ml of 50 mM sodium phosphate buffer containing 500 mM NaCl and 20 mMimidazole, pH 7.6, and sonicated 10 times at a sonication power of 40 Wfor 3 minutes by cycle of a second of ultrasound and a second ofinterruption. The sample obtained, called crude extract, is supplementedwith a final concentration of 2 mM of MgCl₂ and treated for 30 minutesat ambient temperature with DNase I (1 U/ml of crude extract). Theinsoluble cell debris is then removed from the crude extract bycentrifugation for 60 minutes at 20 000 g.

3.2.2 Affinity Chromatography on Nickel Column

The sonication supernatant filtered through a 0.22 μm filter and dilutedto an OD_(280 nm) of 10 is injected onto a HisPrep FF 16/10 affinitycolumn (GC Healthcare®), coupled to the AKTA purifier system (GCHealthcare®), equilibrated in a 50 mM sodium phosphate buffer containing500 mM NaCl and 20 mM imidazole, pH 7.6. The elution is carried out witha gradient of 5% to 30% of a 50 mM sodium phosphate buffer containing500 mM NaCl and 1M imidazole, pH 7.6, at a flow rate of 1 ml/min. Thefractions containing the BOD protein are identified by means of an ABTSactivity test and are combined, concentrated and desalified with a 50 mMsodium phosphate buffer, pH 7.6, by centrifugation on an Amicon YM10membrane. At this stage, the BOD protein is pure and can be stored at−20° C. in soluble form.

Here again, by comparing with the commercially available BODpurification methods, the clear advantage resulting from the use of thisprotein can be emphasized. This is because a single purification step isnecessary in order to obtain a pure enzyme, as opposed to the successionof chromatographies (size exclusion, anion or cation exchange,hydrophobic, etc.) essential for the commercial BODs.

3.2.3 Characterization of Wild-Type BOD Enzymes

3.2.3.1 Molecular Weight Determination

The analysis of the weight of the whole protein was carried out on theLCQ Deca XP mass spectrometer coupled upstream of a nano liquidchromatography apparatus fitted with a C4 desalting andpre-concentrating column (μ-Precolumn™ Cartridge; Acclaim PepMap 300;internal ø 300 μm×5 m; LC Packings Dionex) and of a C4 analytical column(C4 PepMap 300; internal ø 75 μm×5 cm; LC Packings Dionex). A weight of61005.91 Da was obtained for the BOD, i.e. a difference of 130.4 Dacompared with the theoretical weight of the protein; the theoreticalweight is calculated for the protein truncated at the N-terminalmethionine, a difference of only 0.80 Da is found, which demonstratescleavage of this amino acid in the bacterium during the proteinmaturation process.

3.2.3.2 Concentration Measurement

The enzyme concentration of a solution is calculated according to theBradford technique using BSA as standard (Bradford, anal. Biochimie72:248, 1976).

3.2.3.3 Enzymatic Assay

The enzymatic assays are carried out using a Varian spectrophotometer ina 0.1M citrate/phosphate buffer at 37° C. in a volume of 3 ml, with theoxidation of ABTS being followed at 420 nm as a function of time(ε_(420 nm)=36 mM⁻¹ cm⁻¹). The specific activity of the enzyme isexpressed in pmol of ABTS oxidized per minute and per mg of protein. Thestandard ABTS concentration used is 1 mM. The enzyme is diluted so as tomeasure a slope between 0.05 and 0.3 OD_(420 nm)/min.

4. Techniques for Studying the Enzymatic Oroperties of the Wild-TypeBacillus pumilus BOD Enzyme

4.1 Determination of the Kinetic (k_(cat)) and Michaelis (K_(M))Constants in the Stationary State

4.1.1 The Substrate is 2,2′-azinobis(3-ethylbenzothiazoline-6-sulphonicAcid) (ABTS)

The experiments are carried out at 37° C. on a Varian spectrophotometerin a 0.1 M citrate/phosphate buffer, pH 3. The ABTS concentration variesin the test from 0 to 5 mM. The test is triggered by adding enzyme. Theexperimental points are analysed by nonlinear regression according tothe Michaelis-Menten model using the Sigma-plot 6.0 software accordingto the equation below:

Michaelis-Menten model: k _(ss) =k _(cat) *[S]/(K _(M) +[S])

Results:

k _(cat)=391.3 s ⁻¹ and K _(M)=31.7 μM.

FIG. 3 illustrates graphically the specific activity, in U/mg, of theBacillus pumilus BOD as a function of ABTS concentration.

By way of comparison, the homologous CotA protein of Bacillus subtilisexhibits, with respect to ABTS under the same optimal activityconditions, a k_(cat) of 322 s⁻¹ for a K_(M) of 124 μM (Martins et al.,2008. J Biol Inorg Chem, 13:183-193).

4.1.2 The Substrate is Unconjugated Bilirubin

The experiments are carried out at 37° C. in a Varian spectrophotometerin a 50 mM sodium phosphate buffer, pH 7. The bilirubin concentrationvaries in the test from 0 to 60 μM. The test, triggered by the additionof enzyme, consists in following the oxidation of the bilirubin at 450nm by colorimetric change (ε_(450 nm)=32 mM⁻¹ cm⁻¹). The experimentalpoints are analysed by nonlinear regression according to theMichaelis-Menten model using the Sigma-plot 6.0 software according tothe equation below:

Michaelis-Menten model: k _(ss) =k _(cat) *[S]/(K _(M) +[S])

Results:

k _(cat)=70 s ⁻¹ and K _(M)=22 μM.

FIG. 4 is the graphic representation of the Michaelis-Menten equation(k_(ss) in s⁻¹ as a function of unconjugated bilirubin concentration)for the Bacillus pumilus BOD.

4.1.3 The Substrate is Conjugated Bilirubin

The experiments are carried out at 37° C. on a Varian spectrophotometerin a 50 mM sodium phosphate buffer, pH 4.8. The bilirubin concentrationvaries in the test from 0 to 150 μM. The test, triggered by addingenzyme, consists in following the oxidation of the conjugated bilirubinat 440 nm by colorimetric change (ε_(440 nm)=25 mM⁻¹ cm⁻¹). Theexperimental points are analysed by nonlinear regression according tothe Michaelis-Menten model using the Sigma-plot 6.0 software accordingto the equation below:

Michaelis-Menten model: k _(ss) =k _(cat) *[S]/(K _(M) +[S])

Results:

k _(cat)=66.8 s ⁻¹ and K _(M)=35.1 μM.

FIG. 5 represents the catalytic activity for oxidation of the conjugatedbilirubin by the Bacillus pumilus BOD at 37° C. in a 50 mMcitrate/phosphate buffer, pH 4.8.

4.1.4 The Substrate is Syringaldazine (SGZ)

The experiments are carried out at 37° C. on a Varian spectrophotometerin a 50 mM citrate/phosphate buffer, pH 6.2. The SGZ concentration,diluted in methanol, varies in the test from 0 to 300 μM. The test,triggered by adding the enzyme, consists in following the oxidation ofthe SGZ at 530 nm by coloroimetric change (ε_(530 nm)=64 mM⁻¹ cm⁻¹). Theexperimental points are analysed by nonlinear regression according tothe Michaelis-Menten model with competitive inhibition, using theSigma-plot 6.0 software according to the equation below:

Michaelis-Menten model with competitive inhibition:

k _(ss) =k _(cat) *[S]/(K _(M) +[S]+[S] ² /K _(i))

Results:

k _(cat)=116.1; K _(M)=45.6 μM and K _(i)=82.9 μM.

FIG. 6 represents the catalytic activity for oxidation of syringaldazineby the Bacillus pumilus BOD at 37° C. in a 50 mM citrate/phosphatebuffer, pH 6.2.

4.1.5 The Substrate is 2,6-dimethoxyphenol (DMP)

The experiments are carried out at 37° C. on a Varian spectrophotometerin a 50 mM sodium phosphate buffer, pH 6.8. The 2,6-dimethoxyphenolconcentration varies in the test from 0 to 4000 uM. The test, triggeredby adding the enzyme, consists in following the oxidation of the DMP at468 nm by coloroimetric change (ε_(468 nm)=14.8 mM⁻¹ cm⁻¹). Theexperimental points are analysed by nonlinear regression according tothe Michaelis-Menten model using the Sigma-plot 6.0 software accordingto the equation below:

Michaelis-Menten model: k_(ss) =k _(cat) *[S]/(K _(M) +[S])

Results:

k _(ss)=57.3 s ⁻¹ and K _(M)=822 μM.

FIG. 7 represents the catalytic activity for oxidation of DMP by theBacillus pumilus BOD at 37° C. in a 50 mM citrate/phosphate buffer, pH6.8.

4.2 Study as a Function of pH

4.2.1 Activity as a Function of pH

4.2.1.1 ABTS

The study of the variation in the reaction rate constant as a functionof pH is carried out on a pH range of from 3 to 7 in a 0.1 Mcitrate/phosphate buffer, using 1 mM ABTS as substrate. The experimentsare carried out at 37° C. using a Varian spectrophotometer. The activityis followed by oxidation of the ABTS resulting in a colorimetric changemeasured at 420 nm. The test is triggered by adding enzyme.

The results of the oxidation of ABTS, as a function of pH, by theBacillus pumilus BOD are represented as relative activity on the graphof FIG. 8.

4.2.1.2 Unconjugated Bilirubin

The study of the variation in the reaction rate constant as a functionof pH is carried out on a pH range of from 7 to 8.5 in a 0.2 M tris-HClbuffer, using 30 μM unconjugated bilirubin as substrate. The experimentsare carried out at 37° C. using a Varian spectrophotometer. The activityis followed by oxidation of the bilirubin resulting in a colorimetricchange measured at 450 nm (ε_(450 nm)=32 mM⁻¹ cm⁻¹). The test istriggered by adding enzyme.

The results of the oxidation of unconjugated bilirubin, as a function ofpH, by the Bacillus pumilus BOD are represented as relative activity onthe graph of FIG. 8.

4.2.1.3 Conjugated Bilirubin

The study of the variation in the reaction rate constant as a functionof pH is carried out on a pH range of from 3 to 7 in a 0.1 Mcitrate/phosphate buffer, using 100 μM conjugated bilirubin assubstrate. The experiments are carried out at 37° C. using a Varianspectrophotometer. The activity is followed by oxidation of theconjugated bilirubin resulting in a colorimetric change measured at 440nm. The test is triggered by adding enzyme.

The results of the oxidation of conjugated bilirubin, as a function ofpH, by the Bacillus pumilus BOD are represented as relative activity onthe graph of FIG. 8.

4.2.1.4 Syringaldazine (SGZ)

The study of the variation in the reaction rate constant as a functionof pH is carried out on a pH range of from 3 to 7.5 in a 0.1 Mcitrate/phosphate buffer, using 22 μM syringaldazine as substrate. Theexperiments are carried out at 37° C. using a Varian spectrophotometer.The activity is followed by oxidation of the syringaldazine resulting ina colorimetric change measured at 530 nm. The test is triggered byadding enzyme.

The results of the oxidation of syringaldazine, as a function of pH, bythe Bacillus pumilus BOD are represented as relative activity on thegraph of FIG. 8.

4.2.1.5 2,6-Dimethoxyphenol (DMP)

The study of the variation in the reaction rate constant as a functionof pH is carried out on a pH range of from 3 to 7.5 in a 0.1 Mcitrate/phosphate buffer, using 1 mM DMP as substrate. The experimentsare carried out at 37° C. using a Varian spectrophotometer. The activityis followed by oxidation of the DMP resulting in a colorimetric changemeasured at 468 nm. The test is triggered by adding enzyme.

The results of the oxidation of DMP, as a function of pH, by theBacillus pumilus BOD are represented as relative activity on the graphof FIG. 8.

4.2.2 Stability as a Function of pH

The stability as a function of pH, of the wild-type BOD, is determinedby dilution of the enzyme, purified to homogeneity, in a mixed bufferranging from pH 3 to 9 at ambient temperature. This mixed buffer iscomposed of 120 mM Tris, 30 mM imidazole and 30 mM acetic acid, theionic strength of which is adjusted to 190 mM with NaCl. Various samplesare taken as a function of time.

The residual activity is measured at 4° C. using a Varianspectrophotometer, in a 0.1 M citrate/phosphate buffer, pH 3, containing1 mM ABTS.

The results of specific activity and of relative activity of theoxidation of ABTS as a function of pH at 4° C. are represented in thegraphs of FIGS. 9A and 9B.

4.3 Study as a Function of the Temperature

4.3.1 Activity as a Function of Temperature

The study of the variation in the reaction rate constant as a functionof temperature is carried out in a 0.1 M citrate/phosphate buffer, pH 3,in the presence of 1 mM of ABTS.

The temperature ranges from 10 to 85° C. The activity is followed on atemperature-regulated Varian Cary UV Biomelt spectrophotometer. The testis triggered by adding enzyme.

FIG. 10 is a histogram representing the relative activity of theBacillus pumilus BOD as a function of temperature on ABTS oxidation.

4.3.2 Stability of the Enzyme as a Function of Temperature

The enzyme is preincubated at a concentration of 10 mg/ml in a dry bathat 80° C. 2 μl samples are taken and the enzyme is diluted in a 50 mMsodium phosphate buffer, pH 7.6, so as to adjust the enzymeconcentration for the activity test. The residual activity of the enzymeincubated at 80° C. is determined using a Varian spectrophotometer, in a0.1 citrate/phosphate buffer, pH 3, at 37° C., in the presence of 1 mMof ABTS. The test is triggered by adding enzyme.

FIGS. 11A and 11B represent graphically the stability (expressed asspecific activity and as relative activity on ABTS oxidation) of theBacillus pumilus BOD as a function of enzyme incubation time at 80° C.

4.4 Study of the Activity as a Function of the Presence of Urea

The protocol described above in point 4.1.1 was reproduced in thepresence of a urea concentration ranging between 0 and 6 M.

FIGS. 12A and 12B represent graphically the activity (expressed asspecific activity and as relative activity on ABTS oxidation) of theBacillus pumilus BOD as a function of urea concentration at 25° C. andat 37° C.

At 25° C., an activating effect of the urea on the BOD is clearlyobserved. This effect could be due to a slight conformationalmodification of the active site of the enzyme that would be responsiblefor better enzymatic efficiency; this phenomenon, which is known, hasalready been described for other proteins (see Hong-Jie Zhang et al.Biochemical and Biophysical Research Communications 238, 382-386 (1997)and Fan et al. Biochem. J. (1996) 315, 97-102). At 37° C., this effectis not found. It is possible to put forward the hypothesis that thecombined effect of the temperature and of the urea results in too greata modification of the active site, consequently leading to a decrease inthe performance levels of the enzyme.

4.5 Study of the Activity as a Function of the Presence of NaCl

The experiments are carried out at 37° C. on a Varian spectrophotometerin a 50 mM citrate/phosphate buffer, pH 6.2, with increasingconcentrations of NaCl, from 0 mM to 1000 mM. The concentration of SGZ,diluted in methanol, is fixed in the test at 50 μM. The test, triggeredby adding enzyme, consists in following the oxidation of the SGZ at 530nm by colorimetric change (ε_(530 nm)=64 mM ¹⁻.cm ⁻¹) .

FIG. 13 represents the relative activity of SGZ oxidation by theBacillus pumilus BOD as a function of NaCl concentration.

4.6 Study of the Activity as a Function of the Presence of DTT or ofEDTA

The experiments are carried out at 37° C. on a Varian spectrophotometerin a 50 mM citrate/phosphate buffer, pH 6.2, with increasingconcentrations of DTT, from 0 mM to 50 μM, or else of EDTA, from 0 to125 mM. The concentration of SGZ, diluted in methanol, is fixed in thetest at 50 μM. The test, triggered by adding enzyme, consists infollowing the oxidation of the SGZ at 530 nm by colorimetric change(ε_(530 nm)−64 mM⁻¹.cm⁻¹). Table III below collates the resultsobtained, presented in relative activity form.

TABLE III Compound Concentration (mM) Relative activity (%) EDTA 0 100 ±0  0.1 97 ± 1 1 99 ± 5 10 95 ± 1 25 98 ± 3 50 99 ± 4 75 95 ± 2 100 95 ±1 125 89 ± 3 DTT 0 100 ± 0  0.001 99 ± 4 0.005 93 ± 2 0.01 93 ± 4 0.01594 ± 3 0.03 85 ± 1 0.05 81 ± 4

4.7 Study of the Remazol Brilliant Blue R (RBBR) Discoloration Activity

Like many other laccases and bilirubin oxidases, the Bacillus pumilusBOD has a discolouring activity on dyes used in the textile industry.Remazol Brilliant Blue R (RBBR) was selected as an example, and thediscoloration thereof is measured over time in the presence or absenceof a mediator such as ABTS. The experiments are carried out at 37° C. ona Varian spectrophotometer, in a 50 mM potassium phosphate buffer, pH 6,in the absence or presence of ABTS at 3 ml. The RBBR concentration isfixed at 80 mg.l⁻¹ in each tank. The test, triggered by adding 10 μg ofenzyme, consists in following, over time, the discoloration of the RBBRdye at 593 nm.

FIG. 13 represents the discoloration of RBBR by the Bacillus pumilus BODat 3.33 μg.ml⁻¹ at 37° C. in a 50 mM potassium phosphate buffer, pH 6,in the absence or presence of ABTS at 10 μM.

5. Verification of the Presence of the Four Coppers of the Bacilluspumilus Bilirubin Oxidase

The presence of the 4 coppers is determined by means of a bioquinolineassay using a calibration range for copper concentration in order tomeasure the molar concentration of copper (Felsenfeld, G. 1960. Arch.Biochem. Biophys., 87, 247-251; Griffiths et al. 1961, J. Biol. Chem.,236, 1850-1856); the results are given in Table III.

Each measurement, based on a colorimetric assay at 546 nm, is carriedout in duplicate.

This techniques makes it possible to show the presence of 15.3 μM ofcopper for a BOD protein sample at 3.75 μM, i.e. a ratio of 4.08, andclearly confirms the presence of the four copper ions associated withthe enzyme.

Finally, in order to confirm the presence of the 4 coppers in the BODprotein, an elemental analysis on the coppers of the protein was carriedout by atomic absorption. The results clearly confirmed the presence of4 coppers per protein.

TABLE IV Experimental protocol for the bioquinoline assay necessary forassaying the copper of the BOD. Copper Imidazole solution bufferBiquinoline Copper (solu- (solu- (solu- Total concentration tion 2)tion 1) tion 3) volume in the sample Sample (μl) (μl) (μl) (μl) (μM) 1 01200 1800 3000 0 2 0 1200 1800 3000 0 3 120 1080 1800 3000 12.59 4 1201080 1800 3000 12.59 5 240 960 1800 3000 25.18 6 240 960 1800 3000 25.187 360 840 1800 3000 37.77 8 360 840 1800 3000 37.77 9 480 720 1800 300050.36 10 480 720 1800 3000 50.36 11 600 600 1800 3000 62.95 12 600 6001800 3000 62.95 BOD_1 450 750 1800 3000 15.7 (3.75 μm) BOD_2 450 7501800 3000 15.3 (3.75 μM)

1. Purified bilirubin oxidase (BOD), charadterized in that it has apercentage identity of at least 80% with respect to the BOD of Bacilluspumilus of SEQ ID No. 2, in that it catalyses the reaction for oxidationof bilirubin to biliverdin and in that it is bound to four copper atoms.2. Nucleic acid molecule, characterized in that it codes an BODaccording to claim
 1. 3. Nucleic acid molecUle according to claim 2,having a sequence chosen from SEQ ID NO. 1, SEQ ID No. 5 or SEQ ID No.7.
 4. Expression vector, characterized in that it comprises a nucleicacid molecule according to claim
 2. 5. Host cell expressing a BODcharacterized in that it has a percentage identity of at least 80% withrespect to the BOD of Bacillus pumilus of SEQ ID No. 2, in that itcatalyses the reaction for oxidation of bilirubin to biliverdin and inthat it is bound to four copper atoms and further characterized in thatit is transformed with an expression vector according to claim
 4. 6.Method for preparing a BOD characterized in that it has a percentageidentity of at least 80% with respect to the BOD of Bacillus pumilus ofSEQ ID No. 2, in that it catalyses the reaction for oxidation ofbilirubin to biliverdin and in that it is bound to four copper atoms,comprising the steps of: a) preparing host cells according to claim 5;b) culturing host cells prepared in step a); c) lysing said host cells;d) treating the lysate obtained in step c) by affinity chromatography;e) recovering said purified BOD, characterized in that said host cellprepared in step a) is an Escherichia coli BL₂₁ Star strain transformedwith the pFD1 vector and in that said culture carried out in step b) isa liquid-phase culture, with shaking, under anaerobic conditions for aperiod of 20 to 30 h, at a temperature between 20 and 30° C., duringwhich the BOD expression is induced by addingisopropyl-β-D-1-thiogalactopyranoside (IPTG).
 7. Use of the BODaccording to claim 1, for measuring the bilirubin concentration in aliquid sample.
 8. Kit for assaying bilirubin, characterized in that itcomprises a BOD according to claim
 1. 9. Method for assaying thebilirubin in solution in a liquid sample, characterized in that itcomprises the following steps: a) measuring the absorbance atλ_(max)=440 nm of said liquid sample before enzymatic reaction; b)introducing into said liquid sample a BOD according to claim 1; c)measuring the absorbance at λ_(max)=440 nm of said liquid sample afterenzymatic reaction; d) calculating the difference in absorbancesmeasured in steps a) and c) and comparing with differences inabsorbances measured for standard solutions having a known bilirubincontent; e) determining the bilirubin concentration of said liquidsample.
 10. Use of a BOD according to claim 1, for degrading thebilirubin present in a sample.
 11. Use of the BOD according to claim 1,as a reagent in a composition for the oxidation dyeing of keratinfibres.
 12. Use of the BOD according to claim 1, for treating wood pulp.13. Use of the BOD according to claim 1, for discolouring dyes used inindustrial media.
 14. BOD electrode comprising a conductive materialcoated with a deposit comprising at least one BOD according to claim 1.15. Bilirubin biosensor, characterized in that it is constituted of anelectrode according to claim
 14. 16. Oxygen sensor, characterized inthat it is constituted of an electrode according to claim
 14. 17.Enzymatic biofuel cell comprising an anode on which an enzyme catalysingan oxidation reaction is immobilized and an electrode according to claim14 as cathode.